Medical compositions for intravesical treatment of bladder cancer

ABSTRACT

Anti-cancer coating compositions comprising 3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol (E09) are disclosed. More specifically, the coating compositions comprise EO9 and a formulation vehicle. The formulation vehicle improves the solubility and stability of EO9. Additionally, the coating compositions can include coating agents that provide better adhesion of the coating composition to the bladder wall during intravesical delivery of the coating composition.

CROSS REFERENCE To RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/344,446, filed Nov. 1, 2001, and whose entirecontents are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Bladder cancer accounts for approximately 2% of all malignantcancers and is the fifth and tenth most common cancer in men and women,respectively. The American Cancer Society estimated that 54,500 newcases and 11,700 deaths would have occurred in 1997. Superficial bladdercancers (pTa, pT1 and CIS) account for 70-80% of cancers at firstpresentation. Management of superficial bladder cancer may be achievedby endoscopic surgical resection often followed by a course of adjuvantintravesical chemotherapy or immunotherapy with the aim of botheradicating remaining tumor cells and preventing tumor recurrence (HerrH W (1987) Intravesical therapy-a critical review. Urol Clin N Am14:399-404). Both anti-neoplastics (Mitomycin C [MMC], epirubicin andthioTEPA) and immunotherapy (BCG) administered intravesically areeffective at reducing tumor recurrence rates although it is unclearwhether disease progression to muscle invasive tumors is prevented(Newling D (1990) Intravesical therapy in the management of superficialtransitional cell carcinoma of the bladder: the experience of the EORTCGU group, Br J Cancer 61:497-499; Oosterlink et al. (1993) A prospectiveEuropean Organization for Research and Treatment of Cancer GenitourinaryGroup randomized trial comparing transurethral resection followed by asingle instillation of epirubicin or water in single stage Ta, T1papillary carcinoma of the bladder. J Urol 149:749-752). Thisobservation in conjunction with the fact that mortality from bladdercancer is still high underscores the need to develop more effectivetherapeutic agents (Oosterlink et al. 1993).

[0003] One such therapeutic agent is MMC which belongs to a class ofcompounds known as bioreductive drugs (Workman 1994). MMC represents oneof the antineoplastic agents used to treat superficial bladder cancers(Maffezzini et al, 1996, Tolley et al, 1996). MMC is activated to acytotoxic species by cellular reductases although the role of specificreductase enzymes involved in bioreductive activation remains poorlydefined and controversial (Cummings et al, 1998a). This is particularlytrue for the enzyme NQO1 (NAD(P)H:Quinone oxidoreductase, EC 1.6.99.2)which is a cytosolic flavoprotein which catalyses the two electronreduction of various quinone based compounds using either NADH or NADPHas electron donors (Schlager and Powis, 1988, Siegel et al, 1990). Thestructurally related compound E09 (5-aziridinyl-3-hydroxymethyl-1methyl-2-[1H-indole-4,7-dione]prop-(3-en-a-ol), is however a much bettersubstrate for NQO1 than MMC (Walton et al, 1991) and a good correlationexists between NQO1 activity and chemosensitivity in vitro under aerobicconditions (Robertson et al, 1994, Fitzsimmons et al, 1996,Smitkamp-Wilms et al, 1994). Under hypoxic conditions however, EO9'sproperties are markedly different with little or no potentiation of EO9toxicity observed in NQO1 rich cells (Plumb and Workman, 1994). In NQO1deficient cell lines however, large hypoxic cytotoxicity ratios havebeen reported (Workman, 1994). Therefore, EO9 has the potential toexploit the aerobic fraction of NQO1 rich tumors or the hypoxic fractionof NQO1 deficient tumors (Workman, 1994).

[0004] EO9 has been clinically evaluated but despite reports of threepartial remissions in phase I clinical trials, no activity was seenagainst NSCLC, gastric, breast, pancreatic and colon cancers insubsequent phase II trials (Schellens et al, 1994, Dirix et al, 1996).These findings are particularly disappointing in view of the preclinicalstudies (Hendriks et al, 1993) together with reports that several tumortypes have elevated NQ01 levels (Malkinson et al, 1992, Smitkamp-Wilmset al, 1995, Siegel et al, 1998). Several possible explanations havebeen proposed to explain E09's lack of clinical efficacy (Connors, 1996,Phillips et al, 1998). Recent studies have demonstrated that the failureof E09 in the clinic may not be due to poor pharmacodynamic interactionsbut may be the result of poor drug delivery to tumors (Phillips et al,1998). The rapid plasma elimination of E09 (tl/z=10 min in humans) inconjunction with poor penetration through multicell layers suggests thatE09 will not penetrate more than a few microns from a blood vesselwithin its pharmacokinetic lifespan (Schellens et al, 1994, Phillips etal, 1998). Intratumoural administration of E09 to NQ01 rich anddeficient tumors produced significant growth delays (although adistinction between damage to the aerobic or hypoxic fraction was notdetermined) suggesting that if E09 can be delivered to tumors,therapeutic effects may be achieved (Cummings et al, 1998b). While theseundesirable characteristics are a serious setback for the treatment ofsystemic disease, paradoxically they may be advantageous for treatingcancers which arise in a third compartment such as superficial bladdercancer. In this scenario, drug delivery is not problematical via theintravesical route and the penetration of E09 into avascular tissue canbe increased by maintenance of therapeutically relevant drugconcentrations within the bladder (using a one hour instillation periodfor example). While this method of instilling EO9 within the bladder maybe useful, there still remains a need for drug delivery vehicles thatare capable of delivering an effective amount of EO9 within the bladder.

BRIEF SUMMARY OF THE INVENTION

[0005] In a broad aspect, the present invention is directed tocompositions for treating cancer. More specifically, the compositions ofthe present invention comprise pharmaceutical products formulated forintravesical instillation to treat bladder cancer. The pharmaceuticalproducts comprise bioredutive alkylating indoloquinone with anti-tumoreffects such as, but not limited to,3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(E09) and a formulation vehicle. The formulation vehicles of the presentinvention improves the physical characteristics of the solution such assolubility, lyophilization, and ease of reconstitution of thelyophilized solution.

[0006] According to one embodiment of the present invention, thecomposition of the present invention comprises3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(EO9) and a formulation vehicle. According to one embodiment, theformulation vehicle is a mixture of tert-butanol and water. In anotherembodiment, the formulation vehicle is a mixture of ethanol and water.In yet another embodiment, the formulation vehicle is2-hydroxypropyl-β-cyclodextrin. These composition embodiments of thepresent invention can be lyophilized by techniques known or developed inthe art. The lyophilized compositions of the present invention are

[0007] According to another embodiment of the present invention, thecomposition of the present invention comprises EO9 and a coating agent.The coating agent allows for better adhesion of the composition to thebladder wall. Consequently, the composition and, in particular, the EO9contacts and may be able to penetrate the avascular tissue thatcomprises for a time sufficient to treat the bladder cancer. In oneembodiment of the present invention, the coating agent is propyleneglycol. In other exemplary embodiments of the present invention, thecoating agent can be selected from the group consisting ofhydroxypropylcellulose, carboxymethylcellulose, chitosan hydrochloride,lectin, or polycarbophil. In yet another embodiment of the presentinvention, the compositions of the present invention can be delivered tothe bladder wall by a liposome. In another embodiment, the compositionsof the present invention can be delivered to the bladder wall by amicrosphere. In another embodiment, the compositions of the presentinvention can be delivered to a patient intravenously.

BRIEF DESCRIPTION OF DRAWINGS

[0008]FIG. 1. Validation of the polyclonal anti-rat NQO1 antibody foruse in immunohistochemical analysis of human NQO1. Panel A: Western blotanalysis of cell extracts (12.5 p,g protein loaded per lane) for NQO1.Lanes 1-5 represent extracts from DLD-1 (794±121 nmol/min/mg), HT-29(688±52 nmol/min/mg), H460 (1652±142 nmol/min/mg), MT1 (287±53nmol/min/mg), and RT112 (30±3 nmol/min/mg) respectively where the valuesin parenthesis represent NQ01 activity. Lane 6 represents molecularweight markers (ECL protein molecular weight markers, Amersham PharmaciaBiotech, UK). Panel B: Western blot analysis using purified humanrecombinant NQO1. Lanes 1-5 represent protein amounts of 0.25, 0.125,0.0625, 0.0312 and 0.0156 pmol respectively. Panel C: Western blotanalysis of cell extracts (25/,cg protein loaded per lane) derived fromH460 cells (lanes 1-2) and BE cells (lanes 3-4).

[0009]FIG. 2. Immunohistochemical localization of NQ01 in human bladdertumors, normal bladder, urethra and ureter. Tumors (panels A,B and C)were classified as G2 pTa (panel A, [×200]) and G3 pT2 (panels B [×100])and G3 pT4 (panel C [×200]) which had high to intermediate levels ofNQO1 activity as determined by biochemical methods. Panel D (×100)represents a histological section through a macroscopically normallooking section of bladder from a patient who underwent cystectomy for aG3a pT4 tumor; no tumor was identified in these sections but someinflammatory change was evident. Panels E and F (×200) represent urethraand ureter with no evidence of invasive or in situ carcinoma in thesesections. All sections have been stained with NQ01 antibody. Negativestaining (without primary antibody) were clear (data not shown).

[0010]FIG. 3. The relationship between NQO1 activity and the response ofa panel of cell lines to E09 (panel A) or MMC (panel B) under normalphysiological pHe of 7.4 (o) or acidic pHe values of 6.0 ( ). Regressionanalysis data (as determined by Sigma Plot graphics) for E09 at pH 7.4were r=0.886, slope=−0.52 and at pH 6.0, regression analysis data forE09 was r=0.804 and slope=−0.51. For MMC, regression analysis at pH 7.4was r=0.849, slope=−0.19 and at pH 6.0, r=0.609, slope=−0.23.

[0011]FIG. 4. Response of HT-29 multicell spheroids following a one hourexposure to E09 under acidic (pHe=6.0, 0) and physiological (pHe=7.4, 0)extracellular pH conditions. Values presented are the means of 3independent experiments±standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The embodiments of the present invention are directed tocompositions for treating bladder cancer via intravesical instillation.According to one embodiment, the composition of the present inventioncomprises3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol(EO9) and a formulation vehicle. The formulation vehicles of the presentinvention are solvents that improves the solubility and stability ofEO9. In a broad aspect of the present invention, the formulationvehicles of the present invention can be a mixture of an alcohol andwater. According to the various embodiments of the present invention,EO9 dissolves in the formulation vehicles without physical manipulationsuch as grinding. Because the compositions of the present invention arecapable of dissolving greater amounts of EO9, additional flexibilitywith respect to dosage units is achieved. According to one embodiment, acontent of 8.0 mg of EO9 per dosage unit is contemplated. In otherembodiments, instillation doses range from approximately 0.5 mg toapproximately 16 mg in a total volume of 40 mL.

[0013] In addition to improving the solubility of EO9, the formulationvehicles of the present invention are good lyophilization vehicles. Forexample, the formulation vehicles of the present invention minimizes thetime to lyophilize the compositions of the present invention.Accordingly, in one embodiment of the present invention, it is possibleto lyophilize the compositions of the present invention in less thanapproximately 4.5 days. Furthermore, the compositions of the presentinvention are stable after undergoing lyophilization (see table 4). Itis believed that the formulation vehicles of the present inventionminimize the crystallization of EO9 during the lyophilization process.Consequently, by reducing the amount of crystallization of EO9, asmaller volume of fluid is required to reconstitute the compositions ofthe present invention. As a result, a larger batch size can be achieveddue to the reduced reconstitution volumes for the lyophilizedcomposition.

[0014] According to one embodiment, the composition of the presentinvention comprises EO9 and a formulation vehicle comprisingtert-butanol. According to another embodiment of the present invention,the formulation vehicle comprises mixture of ethanol and water. In yetanother embodiment, the formulation vehicle is2-hydroxypropyl-β-cyclodextrin. In one exemplary embodiment, theformulation vehicle comprises 40% tert-butanol in water. As thoseskilled in the art will appreciate, the amount of tert-butanol may bevaried. The tert-butanol solution better dissolves EO9 as compared towater. By utilizing a tert-butanol formulation vehicle, solubility ofEO9 is at least 9.5 mg/ml whereas the solubility of EO9 is approximately0.2 mg/ml in water. Consequently, a smaller volume of the tert-butanolis required to dissolve a given amount of EO9. Additionally, a greateramount of EO9 may be dissolved in a given solution. That is, thecompositions of the present invention will have a higher concentrationof EO9 as compared to a solution where EO9 is dissolved in water.

[0015] According to another embodiment of the present invention, thecomposition comprises, EO9, a formulation vehicle, and a bulking agent.In one exemplary embodiment, lactose can be utilized as the bulkingagent. As those skilled in the art will appreciate, it is contemplatedthat other bulking agents known or developed in the art may be utilized.According to another exemplary embodiment, the composition of thepresent invention can be buffered. In one embodiment, the composition isbuffered to a pH ranging from approximately 9 to approximately 9.5. Thecomposition can be buffered with any known or developed bufferingagents. The compositions of the present invention can either becompounded for intravesical delivery or lyophilized. As those skilled inthe art will appreciate, the compositions of the present invention canbe lyophilized by those methods known or developed in the art. Thelyophilized compositions can be reconstituted by a reconstitutionvehicle. According to one exemplary embodiment, the reconstitutionvehicle comprises 2% sodium bicarbonate, 0.02% disodium edetate andpropylene glycol: water (60:40 V/V). This reconstitution vehicledissolves the lyophilized composition of the present invention andproduces a stable solution for administration for up to 24 hours.Additionally, the reconstitution vehicle of the present inventionprovides an ampoule having an extractable volume of 5 mL ofreconstituted E09 comprising propylene glycol/water/sodiumbicarbonate/sodium edetate 60/40/2/0.02% v/v/w/w.

[0016] In another aspect of the present invention, the compositions ofthe present invention also comprises coating agents. The coating agentsof the present invention provide better adhesion of the composition tothe bladder wall. Consequently, the composition and, in particular, theEO9 contacts and may be able to penetrate the avascular tissue thatcomprises for a time sufficient to treat the bladder cancer. In oneembodiment of the present invention, the coating agent is propyleneglycol. In other exemplary embodiments of the present invention, thecoating agent can be selected from the group consisting ofhydroxypropylcellulose, carboxymethylcellulose, chitosan hydrochloride,lectin, or polycarbophil.

[0017] In yet another embodiment of the present invention, thecompositions of the present invention can be delivered to the bladderwall by a liposome. According to one embodiment of the presentinvention, the liposomes used are unilamellar or multilamellar andcontain at least one cationic phospholipid such as stearylamine,1,2-diacyl-3-trimethylammonium-propane (TAP) or1,2-triacyl-3-dimethylammonium-propane (DAP). In another embodiment ofthe present invention, the surface liposomes may be coated withpolyethylene glycol to prolong the circulating half-life of theliposomes. In yet another embodiment of the present invention, neutrallycharged liposomes such as, but not limited to, phosphatidylcholine andcholesterol can also be used for liposomal entrapment of thecompositions of the present invention. In another embodiment, thecompositions of the present invention can be delivered to the bladderwall by a microsphere such as those known or developed in the art.

[0018] In yet another embodiment, the compositions of the presentinvention can be delivered to a patient intravenously. The lyophilizedcomposition of the present invention can be reconstituted using theformulation vehicles of the present invention. The reconstitutedcomposition can then be diluted to a desired concentration and deliveredto a patient intravenously.

[0019] The following experiments were conducted to determine theactivity of NQ01 in a series of human bladder tumors and normal bladdertissue by both enzymatic and immunohistochemical techniques.Furthermore, the following experiments evaluate strategies for reducingpossible system toxicity arising from intravesical therapy based uponthe fact that the aerobic activity of EO9 against cell lines is enhancedunder mild acidic conditions (Phillips et al., 1992). Administration ofEO9 in an acidic vehicle would result in greater activity within thebladder and any drug absorbed into the blood stream would becomerelatively inactive due to the rise in extracellular pH. The followingexperiments also determine the role of NQO1 in the activation of EO9under acidic conditions.

[0020] Collection of tumor and normal bladder specimens. Ethicalapproval for tissue collection was obtained from the Local ResearchEthical Committee (Bradford NHS Trust) and samples taken from patientsfollowing informed consent. A total of 17 paired cold pinch biopsieswere taken from bladder tumors and macroscopically normal lookingbladder mucosa at cystoscopy, immediately prior to formal transurethralresection of the tumor. Three specimens were taken from patientsundergoing cystectomy and tumor and normal samples dissected bypathologists within one hour of surgical removal. Specimens were flashfrozen in liquid nitrogen and transported for NQOI enzyme analysis.Further biopsies were taken of the normal bladder mucosa immediatelyadjacent to the previous biopsy site and sent at the end of theprocedure, along with the resected tumor, in formalin for routinehistological analysis. In this way bladder tumor and normal bladderurothelium enzymology could be directly correlated with the appropriatetissue histology in each patient. Immunohistochemistry was performedfrom the subsequently archived wax blocks prepared for histology.

[0021] Biochemical determination of NQOI activity. Cell cultures inexponential growth were trypsinised, washed twice with Hanks balancedsalt solution (HBSS) and sonicated on ice (3×30 sec bursts at 40% dutycycle and output setting 4 on a Semat 250 cell sonicator). NQO1 activityand protein concentration was determined as described below. Tissueswere homogenised (10% w/v homogenate) in sucrose (0.25M) using a 1 mltissue homogeniser (Fisher Scientific). Cytosolic fractions wereprepared by centrifugation of the homogenate at 18,000 g for 4 minfollowed by further centrifugation of the supernatant at 110,000 g for 1h at 4° C. in a Beckman Optima TL ultracentrifuge. Activity of NQO1 inthe supernatant was determined spectrophotometrically (Beckman DU650spectrophotometer) by measuring the dicumarol sensitive reduction ofdichlorophenolindophenol (DCPIP, Sigma Aldrich, UK) at 600 nm (Traver etal, 1992). This assay has been extensively validated for use inmeasuring NQO1 activity in both tissue and cell homogenates and has beenshown to be preferable to other assays for NQO1 activity (Hodnick andSartorelli, 1997). Each reaction contained NADH (200 IzM), DCPIP (40/iM,Sigma Aldrich, UK), Dicumarol (20 uM, when required, Sigma Aldrich, UK),cytosolic fraction of tissues (50 p,l per assay) in a final volume of 1ml Tris HCl buffer (50 mM, pH 7.4) containing bovine serum albumin (0.7mg ml⁻¹, Sigma Aldrich, UK). Rates of DCPIP reduction were calculatedfrom the initial linear part of the reaction curve (30 s) and resultswere expressed in terms of nmol DCPIP reduced/min/mg protein using amolar extinction coefficient of 21 mNT′ cm⁻¹ for DCPIP. Proteinconcentration was determined using the Bradford assay (Bradford, 1976).

[0022] Immunohistochemistry. Polyclonal antibodies (raised in rabbits)to purified rat NQO1 were a gift from Professor Richard Knox (EnactPharma Plc). Validation of the antibody for use in immunohistochemistrystudies was performed by Western blot analysis using both purified humanrecombinant NQO1 and cell extracts derived from a panel of cell lines ofhuman origin. These cell lines included H460 (human NSCLC), RT112 (humanbladder carcinoma), HT-29 (human colon carcinoma), BE (human coloncarcinoma), MT1 (human breast) and DLD-1 (human colon carcinoma). The BEcell line has been genotyped for the C609T polymorphic variant of NQOIand is a homozygous mutant (and therefore devoid of NQO1 enzymeactivity) with respect to this polymorphism (Traver et al, 1992). Cellswere washed in ice cold phosphate buffered saline and lysed bysonication (30 seconds on ice) in Tris HCl (50 mM, pH 7.5) containing 2mM EGTA, 2 mM PMSF and 25 Ftg ml⁻¹ leupeptin. Protein concentration wasestimated using the Bradford assay (Bradford, 1976) and a total of 12.5,ug of protein (in Lamelli sample loading buffer) applied to a 12%SDS-PAGE gel. Following electrophoretic transfer to nitrocellulosepaper, membranes were blocked in TBS/Tween 20 (0.1%) containing 5%non-fat dry milk for 1 h at room temperature. Membranes were washed inTBS/Tween 20 (0.1%) prior to the addition of rabbit anti-rat NQO1antibody (1:100 dilution) and incubated at room temperature for 1 h.Membranes were extensively washed in TBS/Tween 20 (0.1%) followed by theaddition of anti-rabbit IgG horseraddish peroxidase conjugated secondaryantibody (1:5000 dilution in TBS/Tween 20). Proteins were visualised byECL based chemiluminescence as described by the manufacturer (AmershamPharmacia Biotech, Bucks, UK).

[0023] For immunohistochemical studies, all tissues (both tumor andnormal bladder mucosa) were fixed in 10% formalin, processed routinelyand embedded in paraffin wax. Two sections of each tissue block wereplaced on one slide, one section served as the test and the other as anegative control (no primary antibody). A total of 5 sections from eachsample were stained for NQ01 (plus negative controls) and tumor andnormal samples from a total of 17 patients were analysed. Sections (5,um) were dewaxed, rehydrated and incubated with primary antibody (1:400dilution) for 4 hours. Sections were then washed and incubated withbiotinylated mouse anti rabbit IgG for 30 min prior to immunoperoxidasestaining using VECTASTAIN ABC reagents and DAB (Vector Laboratories Ltd,Peterborough,UK). Sections were counterstained with haematoxylinaccording to standard procedures.

[0024] Cell culture and chemosensitivity studies. E09 was a gift fromNDDO Oncology, Amsterdam and MMC was obtained from the Department ofPharmacy, St Lukes Hospital, Bradford. H460 (human NSCLC) cell line wasobtained from the American Type Culture Collection (ATCC). HT-29 (humancolon carcinoma), RT112/83 (human bladder carcinoma epithelial), EJ138(human bladder carcinoma) and T24/83 (human bladder transitional cellcarcinoma) cell lines were obtained from the European Collection ofAnimal Cell Cultures (ECACC). A2780 (human ovarian carcinoma) and BE(human colon carcinoma) cells were gifts from Dr T Ward (PatersonInstitute, Manchester, UK). All cell lines were maintained as monolayercultures in RPNII 1640 culture medium supplemented with fetal calf serum(10%), sodium pyruvate (2 mM), L-glutamine (2 mM),penicillin/streptomycin (50 IU/ml/50 jug/ml) and buffered with HEPES (25mK. All cell culture materials were purchased from Gibco BRL (Paisley,UK). Cells were exposed to MMC or E09 at a range of doses for one hourand chemosensitivity was assessed following a five day recovery periodusing the MTT assay, details of which have been described elsewhere(Phillips et al, 1992). The pH of the medium used during drug exposurewas adjusted using small aliquots of concentrated HCl (40 ,A conc HCl[10.5M] to 20 ml medium gives a pH of 6.0). Calibration curves wereconducted over a broad range of pH values in culture medium (pH 3.5 to11) and the stability of the pH conditions monitored over a one hourincubation period at 37° C. At all pH values, no significant changes inthe pH of the medium was observed over the one hour drug exposure period(data not presented).

[0025] HT-29 multicell spheroids were prepared by seeding 5×10⁵ cellsinto T25 flasks which had been based coated with agar (1% w/v) andincubated for 24 h at 37° C. Immature spheroids were then transferred toa spinner flask (Techne) containing 250 ml of RPMI 1640 growth mediumand spheroids were kept in suspension by stirring at 50 rpm. Whenspheroids reached a diameter of approximately 500 Am, they wereharvested for chemosensitivity studies. Multicell spheroids were exposedto a range of E09 concentrations at pHe 6.0 and 7.4 for one hour at 37°C. Following drug incubation, spheroids were washed twice in HBSS priorto dissagregation into single cells using trypsin EDTA. Disaggregatedspheroids were then washed in HBSS and then plated into 96 well plates(1×10³ cells per well). and incubated at 37° C. for four days.Chemosensitivity was assessed using the NM assay as described elsewhere(Phillips et al, 1992).

[0026] The role of NQO1 in the activation of E09 at pHe values of 7.4and 6.0 was evaluated using the NQO1 inhibitor Flavone Acetic Acid(FAA), details of which are described elsewhere (Phillips, 1999). FAA isa competetive inhibitor of NQ01 with respect to NADH and at a finalconcentration of 2 mM, inhibition of NQO1 is >95% whereas the activityof cytochrome P450 reductase and cytochrome b5 reductase is notsubstantially altered (<5% inhibition). Briefly, H460 cells (NQO1 rich)were plated into 96 well plates at a density of 2×10³ cells per well.Following an overnight incubation at 37° C., medium was replaced withfresh medium (pH 7.4) containing a non-toxic concentration of FAA (2 mM)and incubated for one hour at 37° C. Medium was then replaced with freshmedium containing E09 (range of drug concentrations) and FAA (2 mM) ateither pHe 7.4 or 6.0. Following a further one hour incubation at 37°C., cells were washed twice with HBSS and incubated at 37° C. in growthmedium for five days. Chemosensitivity was determined by the NM assay asdescribed above and results were expressed in terms of IC5₀ values,selectivity ratios (IC_(So) at pHe 7.4/IC50 at pHe 6.0) and protectionratios (ICSO FAA/E09 combinations/IC50 for E09 alone).

[0027] Substrate specificity. The influence of acidic pHe on substratespecificity for purified human NQO1 was determined as describedpreviously (Phillips 1996, Walton et al, 1991). NQO1 mediated reductionof the quinone to the hydroquinone species is difficult to detect byconventional assays thereby necessitating the use of a reporter signalgenerating step. In this assay, the hydroquinone acts as an intermediateelectron acceptor which subsequently reduces cytochrome c which canreadily be detected spectrophotometrically. Recombinant human NQ01 wasderived from E. coli transformed with the pKK233-2 expression plasimdcontaining the full length cDNA sequence for human NQ01 isolated fromthe (Beall et al, 1994). Following IPTG induction, NQO1 was purified bycybacron blue affinity chromatography, details of which are describedelsewhere (Phillips, 1996). The purified protein had a molecular weightof approximately 31 kDa and a specific activity of 139/Amol DCPIPreduced/min/mg protein (Phillips, 1996). Reduction of E09 by recombinanthuman NQO1 was determined at pH 6.0 and 7.4 by measuring the rate ofreduction of cytochrome c was measured at 550 nm on a Beckman DU 650spectrophotometer according to previously published methods (Phillips,1996). Results were expressed in terms of ,umol cytochrome creduced/min/mg protein using a molar extinction coefficient of 21.1mM⁻¹cm⁻¹ for cytochrome c.

[0028] Measurement of intracellular pH. Intracellular pH was determinedusing the fluorescent pH indicator BCECF(2,7-bis-(2-carboxy-ethyl)-5-(and-6) carboxyfluorescein (MolecularProbes, Eugene, USA) according to manufacturers instructions. Confluentflasks of cells were washed with HBSS to remove any traces of serumcontaining RPMI medium and then incubated with the esterified form ofBCECF (BCECF-AM) at a concentration of 2 μM in HBSS for one hour at 37°C. The non-denaturing detergent Pluronic was added to the probe to aiddispersion. Cells were then washed to remove all traces of BCECF-AM andthen trypsinized before being suspended in serum-free/phenol red-freeRPM1 medium (Gibco BRL, Paisley, UK) at a concentration of 106 cells perml at pH 6 for one hour. Flourescence measurement was determined in aPerkin-Elmer fluorescence spectrophotometer in UV grade disposable 4 mlcuvettes (Fischer Scientific) with excitation wavelengths 500 nm and 450nm (excitation bandpass slit of 10 nm) and emission wavelength fixed at530 nm (emission bandpass slit of 2.5 nm). These were determined to beoptimal settings for the machine and system under study. An in-situcalibration was performed for every pHi determination with a range ofsix pH's from 4 to 9 using the ionophore nigericin at a concentration of22.8 p,M to equilibrate pHe with pHi. Calculation of the ratio offluorescence at 500 nm/450 nm was calculated after subtraction ofbackground fluorescence from blanks at each pH (serum free, phenol redfree RPMI without cells).

[0029] Activity of NQO1 in tumor and normal bladder specimens. Thebiochemical activity of NQO1 in paired samples of tumor (grade/stageranging from G2 pTa to G2/G3 T4) and normal bladder mucosa (with threecystectomy specimens) taken from a series of 20 patients is presented intable 1. Within the tumor specimens, a broad range of NQO1 activityexisted ranging from 571.4 nmoUmin/mg to undetectable (<0.1nmol/min/mg). In histologically normal bladder mucosa specimens, NQO1activity ranged from 190.9 to <0.1 nmoUmin/mg. In the majority ofpatients NQO1 activity in the tumor was greater than in the normalbladder mucosa. Tumor grade and stage did not correlate with NQO1activity (table 1).

[0030] Validation of NQO1 antibody and immunohistochemical localizationof NQO1. Western blot analysis demonstrates that polyclonal anti ratNQO1 antibody cross reacts with human NQO1 (FIG. 1) with a single bandat approximately 31 kDa observed for both cell extracts and purifiedhuman NQO1. Titration of purified NQO 1 results in a decrease in bandintensity (FIG. 1B) and in cell extracts, band intensity wasqualitatively consistent with NQO1 enzyme activity (FIG. 1A). Inaddition, the antibody does not detect NQO1 in the BE cell line which isdevoid of NQO1 activity as a result of the C609T polymorphism (FIG. 1C).No non-specific bands were observed on Western blots. Immunoperoxidasestaining of NQO1 protein in tumor tissue, bladder wall, ureter andurethra are presented in FIG. 2. Superficial and invasive tumors(pTa-panel A, G3 pT2-panel B and G3pT4-panel C) with high tointermediate levels of NQO1 as determined by biochemical assays (patientnumbers 1, 4 and 5 in table 1) clearly stained positive for NQO1.Staining was confined to the cytoplasm of tumor cells with little or nostaining of stromal cells (panels B and C).

[0031] In other tumors with intermediate or low levels of NQOI activity,staining was heterogeneous with pockets of cells containing high levelsof NQO1 protein (data not shown). Normal bladder wall sections wereobtained from a patient who underwent cystectomy (G3pT4 bladder tumor),ureter and urethra were obtained from another patient who underwentcystectomy (G3 pT3a bladder tumor). In the bladder wall, no NQO1staining was observed in the urothelium (panel D) although slightstaining was present in smooth muscle layers. The urethra (panel E) wasnegative although cells on the luminal surface of the ureter werepositively stained (panel F). The basal layers of the ureter lining werehowever negatively stained (panel F). No evidence of invasive malignancyor in situ carcinoma were observed in the ureter and urethra or in thesection of bladder wall presented (panel D). In 16 other normal bladderbiopsy and cystectomy specimens, no positive staining of the urotheliumwas observed (data not shown).

[0032] Influence of pH on substrate specificity and chemosensitivity.The ability of E09 to serve as a substrate for NQO1 was not influencedby pH with specific activities of 21.10±2.3 and 21.30±1.5 pmolcytochrome c reduced/min/mg protein at pH 7.4 and 6.0 respectively. Theresponse of a panel of cell lines with a range of NQO1 activity (<1.0 to1,898±276 nmol/min/mg) to E09 and MMC at pHe values of 7.4 and 6.0 ispresented in table 2 and FIG. 2. At pHe=7.4, a good correlation existedbetween NQO1 activity and chemosensitivity to E09 (FIG. 3). In the caseof MMC (table 2, FIG. 3), a relationship between NQO1 andchemosensitivity was apparent (at pHe 7.4) although this relationshipwas not as prominent as shown by E09 with a narrow range of IC50 values(range 0.9 to 7.0 ttM) observed in cell lines which cover a broad rangeof NQO1 activity (ranging from <1.0 to 1,898 nmol/min/mg). Both MMC andE09 are preferentially more toxic to cells at pHe values of 6.0 althoughmuch greater potentiation of E09 activity is seen with SR values(SR=selectivity ratio defined as IC₅₀ pHe 7.4_(/IC50) pHe 6.0) rangingfrom 3.92 to 17.21 for E09 compared with 1.02 to 4.50 for MMC (table 2).The activity of E09 was enhanced in both NQO1 rich and deficient celllines when pHe was reduced to 6.0 and the relationship between NQO1 andchemosensitivity remained good when cells were exposed to E09 underacidic conditions (FIG. 3). No cell kill was observed in controlcultures when the pHe was decreased to 6.0 (in the absence of drug) asdetermined by the MTT assay. The response of H460 cells to E09 at pHevalues of 7.4 and 6.0 in the presence and absence of FAA (2 mM) ispresented in table 3. At both pHe values, the response of H460 cells toE09 was reduced in the presence of FAA. Protection ratios defined as theIC50 for E09 plus FAA divided by the IC50 value for E09 alone weresimilar for cells under acidic and physiological pHe values (14.63 and13.95 respectively, table 3). Selectivity ratios defined as the IC50 atpHe 7.4 divided by the IC50 at pHe 6.0 in the presence and absence ofFAA were also similar with SR values of 6.31 and 6.02 for E09 alone andE09 plus FAA respectively (table 3). The response of HT-29 multicellspheroids to E09 is presented in FIG. 4. Spheroids exposed to E09 at pHe6.0 were significantly more responsive than at pHe 7.4 with IC50 valuesof 9.89±0.89 and 24.24±3.29 AM respectively. Spheroids weresignificantly less responsive to E09 than the same cells exposed to E09as monolayers at both pHe values with ratios of IC50 values forspheroids to monolayers of 202 and 341 at pHe values of 7.4 and 6.0respectively.

[0033] Influence of acidic pHe conditions on pHi. PM values following aone hour incubation at pHe 6.0 were 6.44±0.04, 6.51±0.02 and 6.42±0.05in A549, RT112/83 and A2780 cells respectively. Addition of theionophore nigericin (after a one hour incubation at pHe 6.0) resulted inthe equilibration of pHe and p11i.

[0034] In terms of bioreductive drug development, two of the criticalfactors which will ultimately determine selectivity are the enzymologyof tumors and the presence of hypoxia (Workman, 1994). As outlined inthe introduction, the presence or absence of NQO1 is central to thedesign of appropriate E09 based therapeutic strategies aimed attargeting either the aerobic (NQO1 rich cells) or hypoxic fraction (NQO1deficient tumors) of tumors. Workman (1994) has outlined a proposedmechanism for the different properties of E09 under aerobic and hypoxicconditions based on the hypothesis that it is the semiquinone (productof one electron reduction) rather than the hydroquinone which isresponsible for toxicity. In NQO1 deficient cells, the semiquinoneproduced as a result of one electron reductases would be relatively nontoxic as it would rapidly redox cycle back to the parent compound. Freeradical species generated as a result of redox cycling would bedetoxified by superoxide dismutase or catalase but under hypoxicconditions, the semiquinone would be relatively stable. If this were themajor toxic species, then the activity of E09 against cells with lowNQO1 would be potentiated. In NQO1 rich cells however, the major productformed would be the hydroquinone. Aerobic toxicity could be generated asa result of the back oxidation of the hydroquinone to the semiquinonespecies or the parent quinone (Butler et al, 1996) resulting is freeradical generation. Under hypoxic conditions however the hydroquinonewill be more stable and if this is relatively nontoxic, then theactivity of E09 against NQO1 cells under hypoxia would not bepotentiated. Whilst the mechanism of action of E09 under aerobic andhypoxic conditions is complex, the biological data suggest that E09should target the aerobic fraction of NQO1 rich tumors or the hypoxicfraction of NQO1 deficient tumors (Workman, 1994).

[0035] Analysis of NQO1 activity in tumor and normal bladder tissues hasclearly identified patients whose tumors are either NQO1 rich or NQO1deficient (table 1). Within the subset of NQO1 rich tumors, enzymeactivity is elevated relative to the normal bladder urothelium.Immunohistochemical studies confirm these biochemical measurements withstaining confined to tumor cells as opposed to normal stromal cells(FIG. 2, panels A, B and C). Within normal bladder tissues, NQO1staining was absent from the urothelial lining of the bladder (FIG. 2,panel D) and the urethra (FIG. 2, panel E). Faint staining of thesuperficial layers of the ureter (FIG. 2, panel F) was observed althoughthe underlying basal layers of the ureter were negatively stained.Similarly, faint staining of the smooth muscle layers of the bladder,ureter and urethra were also observed (data not shown). These studiessuggest that a proportion of patients with bladder tumors (at variousgrades and stages of the disease) exhibit a significant differential interms of NQO1 activity which could potentially be exploited by E09 basedtherapies directed against the aerobic fraction of tumor cells. Withregards to the ability of E09 to selectively kill hypoxic NQO1 deficientcells, a subset of patients also exist whose tumors are devoid of NQO1activity (table 1). It is not known whether or not bladder tumorscontain regions of low oxygen tension and further studies are requiredusing hypoxia markers such as pimonidazole (Kennedy et al, 1997) toaddress this issue and to establish the relationship between NQO1activity and hypoxia in tumors.

[0036] Whilst biochemical and immunohistochemical studies demonstratethat a subset of patients exist which have the appropriate tumorenzymology to activate E09 (under aerobic conditions), intravesicalchemotherapy can result in systemic toxicity due to the drug enteringthe blood supply. This study has also evaluated a potential strategy forminimizing any risk of systemic toxicity based upon the hypothesis thatadministration of E09 in an acidic vehicle would enhance the potency ofE09 (Phillips et al, 1992) within the bladder and that any drug reachingthe blood stream would become relatively inactive due to a rise in pHe.Selectivity for aerobic cells would still be determined by NQO1 activityand therefore it is essential to determine the role that NQO1 plays inthe activation of E09 under acidic pHe conditions. In a panel of celllines with a broad spectrum of NQO1 activity, reducing the pHe to 6.0enhances the potency of E09 under aerobic conditions in all cases (withSR values ranging from 3.92 to 17.21, table 2). In the case of MMC,potency is also enhanced at low pHe values although the magnitude of thepH dependent increase in toxicity is reduced (SR values ranging from1.02 to 4.50, table 2) compared with E09. With respect to MMC, oneexplanation for increased activity under acidic conditions has beenattributed to the fact that MMC becomes a substrate for NQO1 underacidic conditions (Pan et al, 1993, Siegel et al, 1993). This is not thecase with E09 as rates of reduction of E09 by purified human NQO1 arenot influenced by pH (21.10±2.30 and 21.30±1.50 limol cytochrome creduced/min/mg protein at pH 7.4 and 6.0 respectively). Recent studieshave demonstrated that the activity of E09 is enhanced under acidicconditions (pHe=6.5) but only when the intracellular pH is reduced(plli=6.5) by co-incubation with nigericin (Kuin et al, 1999). Theresults of this study are in agreement with this finding as pHi becomesacidic (pHi values range from 6.42±0.05 to 6.51±0.02 depending on thecell line) when cells are cultured under pHe 6.0 conditions.

[0037] In the panel of cell lines used in this study, a good correlationexists between NQO1 activity and chemosensitivity at both pHe values of7.4 and 6.0 (FIG. 3). A strong relationship between NQO1 activity andresponse under aerobic conditions (at pHe 7.4) has been establishedpreviously by several groups (Robertson et al, 1994, Fitzsimmons et al,1996, Smitkamp-Wilms et al, 1994) and there is clear evidence that NQO1plays a central role in the mechanism of action of E09 under aerobicconditions (Workman, 1994). The good correlation between NQO1 activityand response at pHe 6.0, in conjunction with the fact that E09 is stilla good substrate for NQO1 at pH 6.0, suggests that NQO1 plays asignificant role in E09's mechanism of action at acidic pHe values underaerobic conditions. It is of interest to note however that the activityof E09 against BE cells (which are devoid of NQO1 activity as a resultof the C609T polymorphism, Traver et al, 1992) is also enhanced underacidic pHe conditions (table 2). This suggests that there is a NQO1independent mechanism for the increased activity of E09 under acidicconditions. This is confirmed by the use of the NQO1 inhibitor FAA wherethe ‘protection ratios’ (defined as the ratio of IC₅₀ values for E09plus FAA divided by the ICSo values for E09) are similar at both pHe 7.4and 6.0 (13.95 and 14.63 respectively, table 3). If NQO1 played acentral role in the activation of E09 at pHe 6.0, then the protectionratio at pHe 6.0 would be significantly greater than the protectionratio at pHe 7.4. The mechanism behind the NQO1 independent activationof E09 is unclear although it is a well known fact that the reactivityof aziridine ring structures is enhanced by protonation resulting inring opening to the aziridinium ion which is a potent alkylating species(Mossoba et al, 1985, Gutierrez, 1989). Alternatively, E09 is asubstrate for other one electron reductases (Maliepaard et al, 1995,Saunders et al, 2000) and further studies designed to evaluate whetherE09's metabolism by these enzymes is pH dependent needs to bedetermined. The potency of E09 can be enhanced further by reducing pHebelow 6.0 (Phillips et al, 1992) but these conditions are unlikely toprovide significant clinical benefits as E09 becomes progressively moreunstable when pH is reduced to 5.5 (t'/s=37 min). From a pharmacologicalstandpoint, administration of E09 in a vehicle at pH 6.0 would appeardesirable. Not only would this result in significant enhancement of E09activity but also the stability of E09 would be sufficient (tlh=2.5 h)to maintain drug exposure parameters at a therapeutic level.

[0038] With regards to the activity of E09 against three dimensionalculture models in vitro, this study has demonstrated that reducing thepHe to 6.0 enhances the potency of E09 against multicell spheroidsalthough the magnitude of this effect is reduced compared with monolayercultures (FIG. 4). It is not known whether or not reduction in pHeresults in greater cell kill throughout the spheroid or if it isconfined to the surface of the spheroid exposed to medium. In comparisonwith MMC, previous studies using histocultures exposed to MMCdemonstrated that no difference in toxicity exists between physiologicaland acidic pHe conditions (Yen et al, 1996). The pH dependent increasein E09 toxicity against spheroids suggests that manipulation of pHe maynot only be of use in treating a multilayered solid bladder tumor butmay offer an advantage over MMC. It should however be stated thatmulticell spheroids are significantly less responsive to E09 thanmonolayers, presumably because of the poor penetration properties of E09through avascular tissue (Phillips et al, 1998). E09 can neverthelesskill >90% of cells in spheroids (FIG. 4) suggesting that a higher dosesat least, the penetration of E09 is sufficient to eradicate cells whichreside some distance away from the surface of the spheroid.

[0039] In conclusion, the results of this study have demonstrated thatwithin a population of patients with bladder tumors at various stagesand grades of the disease, there exists a great heterogeneity regardingthe expression of NQO1. The majority of patients have tumors possessingelevated levels of NQO 1 while a small subset of patients appear to bedevoid of NQO1 activity. The heterogeneous nature of NQO1 activitydescribed here is consistent with several other studies in various tumortypes (Malkinson et al, 1992, Smitkamp-Wilms et al, 1995, Siegel et al,1998). These findings reinforce the view that ‘enzyme profiling’ ofindividual patients could be valuable prior to therapeutic interventionwith bioreductive drugs (Workman, 1994). This is to our knowledge thefirst study to characterize NQO1 activity and cellular localization inbladder tumors and provide strong evidence to support the evaluation ofE09 against superficial and locally invasive bladder tumors. This studyhas clearly demonstrated that under aerobic conditions, E09 is much morepotent under acid conditions (pH 6.0) than at physiological pH (pH 7.4).The mechanism for this increased E09 potency appears to be NQ01independent and whilst this will not improve (or reduce) selectivity, itmay prove beneficial in terms of reducing the therapeutically effectivedose of E09. Dose reduction in conjunction with the fact that areduction in the potency of E09 due to the increased pHe in the bloodstream suggests that systemic toxicity arising from the intravesicaladministration of E09 would be low. In addition, this study shows thatunder physiological conditions the activity of E09 is much lower intissues with “normal” expression of NQO1 compared to “high” NQO1expressing tissues (i.e. the tumors). The results of this study providestrong evidence in support of the proposal that intravesicaladministration of E09 may have activity against bladder tumors.

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[0076] Yen W C, Schmittgen T and Au J L (1996) Different pH dependencyof mitomycin C activity in monolayer and three dimensional cultures.Pharmaceut Res 13: 1887-1891. TABLE 1 Tumor histology reports and NQO1activity in paired samples of bladder tumor and normal bladder mucosa.NQO1 Activity Ratio of NQO1 Activity Normal NQO1 levels Patient TumorTumor (nmol/ in tumor to No. histology (nmol/min/mg) min/mg) normaltissue.  1 ^(f,s,i,p) G2 pTa 571.4 <0.1 571.  2 ^(m,s,r) G3 pT2 273.3<0.1 273.  3 ^(f,s,i) G1pTa 107.80 <0.1 107.  4 ^(m,e,i) G3 pT2/3 73.36<0.1 73.3  5 ^(m,s,i) G3pT4 (0′ 81.30 4.10 19.8  6 ^(h) G2PT1 309.5025.20 12.1  7 ^(m,n,r,o) G3 pT2 10.00 <0.1 10.0  8 ^(fn,i) G3pT2 9.80<0.1 9.80  9 m,n,i G2 pT2 4.40 <0.1 4.40 10 m,s,c G3 pT2 34.01 8.50 4.0011 ^(m,s) G 1 pTa 69.76 22.20 3.14 12 ,,n G1pTa 42.16 15.30 2.73 13m,n,i G3 pT2 179.6 72.12 2.49 14 m,e,i G2/G3 T4 (C) 89.70 63.30 1.41 15m,n,r G3 pT2 0.40 <0.1 0.40 16 m,e,c,o G3 PT3 (C) 21.60 61.70 0.35 17 fn,i G2 PTI 58.40 190.90 0.30 18 m,e,o G2 PTI <0.1 <0.1 0 19 f n,i G2PTI. <0.1 <0.1 0 20 m,e,c,r G2 pT0 <0.1 <0.1 0 #carcinogen exposure(i.e., dye industry worker). (C) denotes cystectomy specimens. In allcases, protein levels following preparation of the cytosolic fractionwere greater than 0.1 mg/ml.

[0077] TABLE 2 The relationship between NQ01 activity andchemosensitivity to E09 and MMC under physiological and acidic pHeconditions. IC50 pHe IC50 pHe NQ01 7.5 6.0 Cell line Drug (nmol/min/mg)(nM) (nM) SR* H460 E09 1652 ± 142  60 ± 10 9.5 ± 2   6.31 HT-29 E09 −688 ± 52 120 ± 53 29 ± 10 4.13 T24/83 E09 285 ± 28 290 ± 65 60 ± 18 4.83A2780 E09 159 ± 33 200 ± 50 51 ± 14 3.92 EJ138 E09  83 ± 14 310 ± 95 39± 7  7.94 RT112 E09 30 ± 3 1050 ± 75  61 ± 13 17.21 BE E09 <0.1 5300 ±169 1300 ± 75−    4.07 H460 MMC 1652 ± 142  900 ± 200 220 ± 130 4.50HT-29 MMC 688 ± 52 1050 ± 210 500 ± 240 2.10 T24/83 MMC 285 ± 28 2150 ±93  2100 ± 800  1.02 A2780 MMC 159 ± 33 2400 ± 340 1400 ± 130  1.71EJ138 MMC  83 ± 14 1600 ± 200 1400 ± 250  1.14 RT112 MMC 30 ± 3 3350 ±250 2000 ± 500  1.67 BE MMC <0.1 7000 ± 192 4400 ± 215  1.59

[0078] TABLE 3 Response of H460 cells to E09 in the presence or absenceof FAA (2 mm) at pHe values of 7.4 and 6.0. ICso Drug pHe (nM) SR* PR**E09 7.4 60.0 ± 8.1 ′ — E09 6.0  9.5 ± 2.6 6.31 — E09/FAA 7.4 837 ± 45 —13.95 E09/FAA 6.0− 139 ± 27 6.02 14.63

[0079] TABLE 4 Neoquin 8 mg/vial lyophilised product time (months)Storage test item 0 1 2 3 6  5° C. content* 102.7 ± 1.2  na na 103.8 ±0.8  100.6 ± 0.6  purity**  99.9 ± 0.008 na na 99.5 ± 0.03 99.6 ± 0.03residual 6.0% na na 7.0% 6.3% moisture*** pH after 9.5    na na na9.4    reconstitution**** 25° C./60% content 102.7 ± 1.2  103.4 ± 0.7 102.1 ± 0.2  102.6 ± 1.3  97.4 ± 1.0  RH purity  99.9 ± 0.008 99.9 ±0.05 99.9 ± 0.01 99.2 ± 0.07 98.7 ± 0.2 residual moisture 6.0% na na5.9% 5.9% pH after 9.5    na na na 9.4    reconstitution**** 40° C./75%content 102.7 ± 1.2  102.3 ± 1.1  100.4 ± 1.3  101.3 ± 0.2  86.4 ± 2.0 RH purity  99.9 ± 0.008 99.8 ± 0.01 99.7 ± 0.04 98.4 ± 0.07 97.5 ± 0.2 residual moisture 6.0% na na 6.2% 6.3% pH after 9.5    na na na 9.5   reconstitution****

What is claimed is:
 1. An anti-cancer formulation comprising: a bufferedsolution comprising3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenoland a formulation vehicle.
 2. The anti-cancer formulation of claim 1wherein the anti-cancer formulation is lyophilized.
 3. The anti-cancerformulation of claim 1 wherein the buffered solution has a pH rangingfrom approximately 9 to approximately 9.5.
 4. The anti-cancerformulation of claim 1 wherein the formulation vehicle is selected fromthe group consisting of tert-butanol/water, ethanol/water, and2-hydroxypropyl-β-cyclodextrin solution.
 5. The anti-cancer formulationof claim 1 further comprising a bulking agent.
 6. The anti-cancerformulation of claim 5 wherein the bulking agent is lactose.
 7. Aanti-cancer formulation for treating bladder cancer, the formulationcomprising: a buffered, lyophilized solution comprising3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenoland a formulation vehicle.
 8. The anti-cancer formulation of claim 7wherein the buffered, lyophilized solution has a pH ranging fromapproximately 9 to approximately 9.5.
 9. The anti-cancer formulation ofclaim 7 wherein the formulation vehicle is selected from the groupconsisting of tert-butanol/water, ethanol/water, and2-hydroxypropyl-β-cyclodextrin solution.
 10. The anti-cancer formulationof claim 7 further comprising a bulking agent.
 11. The anti-cancerformulation of claim 10 wherein the bulking agent is lactose.
 12. Ananti-cancer formulation comprising:3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenoldissolved in a buffered formulation vehicle, the buffered formulationvehicle having a pH ranging from approximately 9 to approximately 9.5,and wherein the formulation vehicle is selected from the groupconsisting of tert-butanol/water, ethanol/water, and2-hydroxypropyl-β-cyclodextrin solution.
 13. The anti-cancer formulationof claim 12 further comprising a bulking agent.
 14. The anti-cancerformulation of claim 13 wherein the bulking agent is lactose.
 15. Ananti-cancer formulation comprising: an indolequinone having a pH withina range of approximately 9 and approximately 9.5, a refractive indexwithin a range of approximately 1.393 to approximately 1.406, and arelative density of within a range of approximately 0.94 toapproximately 0.95.
 16. The anti-cancer formulation of claim 15 whereinthe indolequinone is3-hydroxymethyl-5-aziridinyl-1-1-methyl-2-[1H-indole-4,7-dione]propenol.17. The anti-cancer formulation of claim 16 further comprising a coatingcomposition.
 18. The anti-cancer formulation of claim 17 wherein thecoating composition is selected from the group consisting of propyleneglycol, hydroxypropylcellulose, carboxymethylcellulose, chitosanhydrochloride, lectin, and polycarbophil.